Rotavirus (RV) is a leading global cause of childhood diarrhea. Current attenuated human RV (AttHRV) oral vaccines are effective in infants in developed countries, but for unexplained reasons, they lack efficacy in impoverished countries where diarrhea mortality is highest. Alternative affordable strategies are urgently needed to reduce diarrheal diseases and enhance oral vaccine efficacy. Malnutrition (kwashiorkor) and frequent antibiotic usage in infants affect the gut microbiome and barrier integrity, likely compromising gut immunity and predisposing infants to diarrheal illness, but their impact on HRV infection is uncharacterized. Studies of gnotobiotic (Gn) animals have revealed that probiotics or commensal microflora are crucial for gut immune maturation and homeostasis, but the mechanisms involved and their roles in modulating viral diarrheas or enhancing oral vaccine efficacy are unclear. Neonates undergo progressive changes in the gut microflora which we will model by colonizing Gn pigs with Lactobacillus spp (Gram, G+), E. coli (G- ), both, or a commensal cocktail from conventional piglets. Gn pigs resemble infants in size, anatomy, physiology, development of mucosal immunity and are the only animal model susceptible to HRV diarrhea. Our investigation of how the selected probiotics/commensals modulate gut homeostasis and immune responses and impact enteric diseases and vaccines will permit their rational use as biotherapeutic agents and/or adjuvants. We hypothesize that selected G+ and G- gut microflora will modulate different host cellular pathways leading to immunostimulatory, but balanced (Th1/Th2/Th17/Treg) responses that enhance efficacy of RV vaccines or immuno-regulatory (Treg) responses that moderate HRV diarrhea. Further, humanized, outbred Gn pigs are a unique model to study how microbiota and diet contribute to malnutrition and HRV disease severity under conditions that constrain confounding variables in ways not possible in infants. In Aim 1, we determine how selected G+ or G- probiotics, both, or the commensal cocktail modulates immune responses, gut homeostasis and HRV pathogenicity. Then we test the impact of antibiotics on the commensal microflora and on these same parameters. In Aim 2, we determine how defined probiotics or commensals modulate protective immunity to AttHRV oral vaccine. In Aim 3, in collaboration with Jeff Gordon, we develop a humanized Gn pig model to study the interaction of microbiota x diet (from African twins discordant for kwashiorkor) on severe malnutrition and HRV pathogenicity and identify microbial biomarkers to aid in vaccine design or therapeutic interventions. Effects of the probiotics/commensals/microbiota will be compared by intestinal transcriptome profiling (pig microarrays, 44K ESTs), metagenomics, metabolomics and metatranscriptomics, gut barrier integrity (sugar permeability, tight junction genes, serum LPS), and induction of innate and adaptive immune responses, to establish the immunoregulatory/immunostimulatory profiles. Our innovative studies will address gaps in knowledge of gut immune maturation and homeostasis and interactions between gut microflora and enteropathogenic viruses or oral vaccines. Our findings will contribute to alternative low cost probiotic treatments applicable to infants (or mothers) to moderate HRV disease, enhance oral vaccine efficacy, and reduce infant morbidity and mortality.